With advancing age the mammalian thymus undergoes involution, a progressive loss of architectural integrity and lymphoid cellularity, that results in reduced T lymphopoiesis. Thymic involution is frequently associated with states of immune deficiency, such as active HIV infection, or severe malnutrition as well as advanced age. Immune recovery appears to require restoration of normal thymopoiesis. While a number of means are known to increase overall T cell differentiation, there has been a paucity of treatments that target the thymus while having no effect on the peripheral immune system. Our goal here is to develop a customized gene therapy delivery system targeting the thymus that can be employed to slow or reverse the thymic involution process. Intrathymic T cell development requires mutual, two-way interactions between developing lymphoid cell and the thymic stroma. T cell progenitors enter the thymic medulla from the blood and undergo directed migration through the cortex and the medulla, receiving key differentiative cues from unique intrathymic stromal microenvironments. With advancing age the thymic architecture and cellular composition change, as does stromal gene expression. The thymic epithelial stroma will be the target of our gene therapy approach. The first specific aim is to optimize a gene therapy delivery system based on the novel approach developed in our lab: intrathymic injection of engineered cell lines that integrate into the thymic structure and express the gene product of interest locally. Several cell lines and promoters will be tested for the most effective vector construction, with the goal of developing a cell delivery vehicle that integrates into the thymic architecture, produces high levels of the therapeutic protein over an extended period and does not otherwise disrupt thymic structure or T cell development. The second specific aim will use this gene delivery method to target the aged thymic epithelium. Implanted cells will be engineered to express either the morphogen Wnt4 or keratinocyte growth factor, both proteins that affect stromal cell function. These studies will determine whether thymic epithelial function can be enhanced by a local increase in either of these proteins and thereby prevent or reverse the involution process. This delivery system can be employed to assess the potential of any number of other therapeutic proteins, to achieve the ultimate goal of increasing thymic T cell output and thereby improving immune function in the elderly.